The present invention relates to penta-saccharides, to processes for their preparation and to pharmaceutical compositions containing them.
Heparin is a polysaccharide of the glycosamino-glycan family which is known for its anticoagulant properties. It is known (I. Bjxc3x6rk and U. Lindahl, Molecular and Cell Biochemistry, 1982, Dr. W. Junk Publishers-Netherlands) that blood clotting is a complex physiological phenomenon. Certain stimuli, such as contact activation and tissue factor, trigger the subsequent activation of a series of clotting factors present in blood plasma. Irrespective of the nature of the stimulus, the final steps are identical, the activated factor X (Xa) activates factor II (also known as prothrombin), which, in its activated form (factor IIa, also known as thrombin), gives rise to partial proteolysis of soluble fibrinogen with release of insoluble fibrin, which is one of the main constituents of a blood clot.
Under normal physiological conditions, the activity of the clotting factors is regulated by proteins such as antithrombin III (ATIII) and heparin cofactor II (HC II), which are also present in plasma. AT III exerts inhibitory activity on a certain number of clotting factors, and in particular on factors Xa and IIa.
Inhibition of factor Xa or of factor IIa is thus the preferred means for obtaining anticoagulant and antithrombotic activity, since these two factors are involved in the final two steps of clotting, which are independent of the triggering stimulus.
A pentasaccharide such as the one described by P. Sinay et al., Carbohydrate Research 1984, 132 C5 represents the minimum heparin sequence required for binding to AT III. This compound was obtained about fifteen years ago by total chemical synthesis.
Since then, a certain number of synthetic oligosaccharides, obtained by total chemical synthesis and having antithrombotic anticoagulant activity, have been described in the literature.
Patent EP 0,084,999 describes derivatives consisting of uronic acid (glucuronic or iduronic acid) monosaccharide units and glucosamine which have advantageous antithrombotic properties. Besides substituents consisting of hydroxyl groups, these compounds contain N-sulphate groups, N-acetyl groups and, in certain cases, the anomeric hydroxyl groups are replaced with methoxy groups.
Application EP 0,165,134 also describes synthetic oligosaccharides with antithrombotic activity. These compounds, consisting of uronic acid monosaccharide units and glucosamine and containing an O-sulphate group in position 3 of the glucosamine unit, are also described in application EP 0,301,618. These compounds have powerful antithrombotic and anti-coagulant properties. Patent EP 0,454,220 describes uronic acid derivatives and glucose derivatives which contain O-alkyl or O-sulphate groups as substituents. These latter compounds also have antithrombotic and anticoagulant properties.
Sulphated glycosaminoglycanoid derivatives in which the N-sulphate, N-acetate or hydroxyl functional groups have been replaced with alkoxy, aryloxy, aralkyloxy or O-sulphate groups are also described in patent EP 0,529,175. These compounds have advantageous antithrombotic properties. The latter compounds are also inhibitors of smooth muscle cell proliferation.
Oligosaccharides, and in particular penta-saccharides, which are analogous to the minimum heparin sequence required for binding to AT-III are described in Angew. Chem. Int. Ed. Engl. 1993, 32, 3, 434-436. These compounds contain glucuronic acid or glucose units whose hydroxyl functions have been replaced with O-sulphate or O-methyl groups.
Many studies have since been carried out on pentasaccharides, and it has been indicated in the literature that the conformation of the L-iduronic acid unit G plays an important role in the activity of the products. Several conformational states for the unit G have been described (4C1, 1C4, 2S0) and it has been suggested that this conformational flexibility is essential for the biological activity of products containing L-iduronic acid (B. Casu, M. Petitou, A. Provasoli and P. Sinay, Conformational flexibility: a new concept for explaining binding and biological properties of iduronic acid-containing glycosamino-glycans. Trends Biochem. Sci. 1988, 13, 221-225).
It has now been found, surprisingly, that by replacing one of the O-alkyl groups with an alkylene bridge, and thus by locking the conformation of the L-iduronic acid, oligosaccharides are obtained which have advantageous biological properties although lacking in conformational flexibility. The reason for this is that the compounds of the present invention differ from the other synthetic heparinoids described in the literature, by virtue of their novel structures and their powerful and unexpected biological properties. The compounds of the invention are pentasaccharides in which the L-iduronic unit G is in the so-called xe2x80x9clockedxe2x80x9d2S0 conformation and in which the D-glucuronic acid unit E optionally has an ethyl group in position 5. These compounds have very great anti-factor Xa activity and great affinity for AT III.
The subject of the present invention is, more particularly, a pentasaccharide in acidic, form and its pharmaceutically acceptable salts, with one or more pharmaceutically acceptable cations, the anionic form of which has the formula (I): 
in which:
R represents hydrogen or an xe2x80x94SO3xe2x88x92, (C1-C3)alkyl or (C2-C3)acyl group;
T represents hydrogen or an ethyl group; and
n represents 1 or 2.
The invention encompasses pentasaccharides in acidic form, or in the form of a pharmaceutically acceptable salt. In the acidic form, the xe2x80x94COO and xe2x80x94SO3 functions are in the form xe2x80x94COOH and xe2x80x94SO3H, respectively.
The expression xe2x80x9cpharmaceutically acceptable salt of the pentasaccharides of the inventionxe2x80x9d is intended to refer to pentasaccharides in which one or more of the xe2x80x94COO and/or xe2x80x94SO3 functions are ionically bonded to a pharmaceutically acceptable metal cation.
The preferred salts according to the invention are those in which the cation is chosen from the cations of alkali metals and, even more preferably, those in which the cation is Na+ or K+.
The subject of the present invention is also a process for preparing the pentasaccharides of the invention, characterized in that a precursor of the unit G is prepared, which is coupled with a precursor of the unit H to give a precursor of GH, the pentasaccharide finally being obtained: either by coupling a precursor of GH with a precursor of DEF, or by coupling a precursor of GH with a precursor of EF, followed by addition of D.
Any precusor of G, of H, of EF or of DEF can be used. This means that it is possible, according to s these processes, to prepare an entire family of pentasaccharides having the unit G of locked configuration in common.
The process described above is the preferred process of the invention. However, the pentasaccharides of the invention can be prepared by other known methods of sugar chemistry, and in particular by reacting a monosaccharide containing protective groups such as described by T. W. Green, in Protective Groups in Organic Synthesis (Wiley, N.Y. 1981), on the hydroxyl radicals and optionally on the carboxyl radicals, if present, with another protected monosaccharide, to form a disaccharide which is then reacted with another protected monosaccharide to form a protected trisaccharide, from which a protected tetrasaccharide and then a protected pentasaccharide can be obtained.
The protected pentasaccharides are then deprotected and optionally sulphated, or partially deprotected, then sulphated and then deprotected, in order to obtain the compounds of the invention.
Such processes are known in carbohydrate chemistry, and are described in particular by G. Jaurand et al. in Bioorganic and Medicinal Chemistry Letters 1992, 2, 9, 897-900, by J. Basten et al., in Bioorganic and Medicinal Chemistry Letters 1992, 2, 9, 905-910 and by M. Petitou and C. A. A. van Boeckel in xe2x80x9cChemical synthesis of heparin fragment and analoguesxe2x80x9d 203-210xe2x80x94Process in the chemistry of organic natural products, Ed. Springer Verlag Viennaxe2x80x94N.Y. 1992.
The process described above makes it possible to obtain the compounds of the invention in the form of salts. In order to obtain the corresponding acids, the compounds of the invention in the form of salts are placed in contact with a cation-exchange resin in acidic form.
The compounds of the invention in the form of acids can then be neutralized with a base in order to obtain a desired salt.
To do this, any inorganic or organic base which gives pharmaceutically acceptable salts can be used.
Sodium hydroxide, potassium hydroxide, calcium hydroxide or magnesium hydroxide is preferably used. The sodium and calcium salts of the pentasaccharides of the invention are the preferred salts.
The compounds which are the subject of the present invention have advantageous pharmacological and biochemical properties. More particularly, they have great anti-factor Xa activity and great affinity for AT III.
As has been mentioned above, in its clotting cascade, factor Xa activates prothrombin into thrombin, which proteolyses soluble fibrinogen with release of insoluble fibrin, the main constituent of a blood clot.
Inhibition of factor Xa is thus a preferred means for obtaining anticoagulant and antithrombotic activity. The anti-factor Xa activity of the products of the invention was evaluated at pH 7, according to the method described by Teien A. N. and Lie M., in Thrombosis Research 1977, 10, 399-410, and it has been demonstrated that the products of the invention have anti-Xa activity equal to or greater than that of the synthetic heparinoids already known.
The affinity of the pentasaccharides of the invention, the anion of which has the formula (I), for AT III was determined by spectrofluorimetry, under the conditions described by D. Atha et al. in Biochemistry 1987, 26, 6454-6461. The results of the tests demonstrated that the compounds of the invention have very great affinity for AT III.
Moreover, the overall antithrombotic activity of these compounds was evaluated in rats by means of a model of venous stasis and induction with thrombo-plastin, according to the method described by J. Reyers et al. in Thrombosis Research 1980, 18, 669-674.
The ED50 of the compounds of the invention is at least of the same order as or less than that of the other synthetic heparinoids already known. The compounds of the invention thus have advantageous specificity of action and advantageous anticoagulant and antithrombotic activity.
The compounds of the invention are useful for preparing pharmaceutical compositions for parenteral administration.
The compounds of the invention are of very low toxicity; their toxicity is entirely compatible with their use as medicines.
The compounds of the invention are very stable and are thus particularly suitable to be the active principle of medicines.
The invention also covers pharmaceutical compositions containing, as active principle, a compound according to the invention or one of its pharmaceutically acceptable salts, optionally combined with one or more inert and appropriate excipients.
In each unit dose, the active principle is present in the amounts which are suited to the daily doses envisaged. Each unit dose contains from 0.1 to 100 mg of active principle, preferably from 0.5 to 50 mg.
The compounds according to the invention can also be used in combination with one or more other active principles which are useful for the desired therapy, such as, for example, antithrombotic agents, anticoagulants, anti-platelet-aggregating agents such as, for example, dipyridamole, aspirin, ticlopidine, clopidogrel or antagonists of the IIb/IIIa glycoprotein complex.
The pharmaceutical compositions are formulated for administration to mammals, including man, for the treatment of the abovementioned diseases.
Pharmaceutical compositions thus obtained are advantageously in various forms such as, for example, injectable or drinkable solutions, sugar-coated tablets, plain tablets or gelatin capsules. The injectable solutions are the preferred pharmaceutical forms. The pharmaceutical compositions of the present invention are useful in particular for the preventive or curative treatment of vascular wall disorders, such as atherosclerosis, hypercoagulability states observed, for example, after tumour surgery or disruption of clotting, induced by bacterial, viral or enzymatic activators.
More generally, the pentasaccharides of the invention can be used in the treatment of pathologies associated with a clotting dysfunction.
The dosage can vary within a wide range depending on the patient""s age, weight and state of health, the nature and severity of the complaint and the route of administration. This dosage comprises the administration of one or more doses of from about 0.5 mg to about 1000 mg per day, preferably from about 1 to about 100 mg per day and better still from about 0.5 to about 50 mg per day, for example about 20 mg per day, intramuscularly or subcutaneously in batchwise administrations or administrations at regular intervals, or of a daily dose of about 200 mg to about 1000 mg per day orally.
Naturally, these doses can be adjusted for each patient, depending on the results observed and on the blood analyses carried out beforehand.
Subcutaneous administration is the preferred route
The subject of the present invention is thus also pharmaceutical compositions which contain, as active principle, one of the above compounds optionally combined with another active principle. These compositions are made so as to be able to be administered digestively or parenterally.
In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, transmucous, local or rectal administration, the active ingredient can be administered in unit forms of administration, mixed with standard pharmaceutical supports, to animals and to human beings. The appropriate unit forms of administration comprise oral forms such as oral suspensions, solutions, granules and powders, gelatin capsules and tablets, sublingual and buccal forms of administration, subcutaneous, intramuscular, intravenous, intranasal or intraocular forms of administration and rectal forms of administration.
When a solid composition is prepared in the form of tablets, the main active ingredient is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose or other suitable materials or alternatively they can be treated such that they have sustained or delayed activity and so that they continuously release a predetermined amount of active principle.
A preparation as gelatin capsules is obtained by mixing the active ingredient with a diluent and pouring the mixture obtained into soft or hard gelatin capsules.
The water-dispersible powders or granules can contain the active ingredient mixed with dispersing agents or wetting agents, or suspending agents, such as polyvinylpyrrolidone, as well as with sweeteners or flavour enhancers.
For rectal administration, use is made of suppositories which are prepared with binders which melt at rectal temperature, for example cocoa butter or polyethylene glycols.
For parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically compatible dispersing agents and/or wetting agents, for example propylene glycol or butylene glycol, are used.
For transmucous administration, the active principle can be formulated in the presence of a promoter such as a bile salt, a hydrophilic polymer such as, for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, pectins, starches, gelatin, casein, acrylic acids, acrylic esters and copolymers thereof, vinyl polymers or copolymers, vinyl alcohols, alkoxy polymers, polyethylene oxide polymers, polyethers or a mixture thereof.
The active principle can also be formulated in the form of microcapsules, optionally with one or more supports or additives.
The active principle can also be in the form of a complex with a cyclodextrin, for example xcex1-, xcex2- or xcex3-cyclodextrin, 2-hydroxypropyl-xcex2-cyclodextrin or methyl-xcex2-cyclodextrin.
The active principle can also be released by a balloon containing it or by an endovascular extender introduced into the blood vessels. The pharmacological efficacy of the active principle is thus not affected.
Subcutaneous administration is the preferred route.
The methods, the preparations and the schemes which follow illustrate the synthesis of the various intermediates which are useful for obtaining the pentasaccharides according to the invention.
The following abbreviations are used: TBDMS: tert-butyldimethylsilyl; Lev: levulinyl; Bn: benzyl; Bz: benzoyl; TLC: thin layer chromatography; Olm: trichloroacetimidyl; LSIMS: liquid secondary ion mass spectrometry; ESIMS: electron spray ionization mass spectrometry; TMS: trimethylsilyl; TSP: sodium trimethylsilyltetradeuteropropionate; Tf: triflate; MS: molecular sieves; All: allyl; PMB: p-methoxybenzyl; SE: trimethylsilylethyl. Dowex(copyright), Sephadex(copyright), Chelex(copyright) and Toyopearl(copyright) are registered trade marks.
In the methods, the preparations and the examples described below, the general procedures relating to the catalytic coupling of the imidates, the cleavage of the levulinic esters, the catalytic coupling of the thioglycosides, the saponification, methylation and selective deprotection of the p-methoxybenzyl group, the deprotection and sulphation of the oligo- and polysaccharides by hydrogenoloysis of the benzyl esters or ethers, the saponification of the esters or the sulphations can be carried out by applying the general methods below to the appropriate intermediates.